Publicly available genetic sequence data were searched for human sequences that potentially represent protein kinases, important players in virtually every signaling pathway. After removal of duplicates, splice variants and pseudogenes, this search yielded 510 sequences with recognizable similarity to eukaryotic protein kinases.

Background Protein kinase C (PKC) has become a major focus among cell biologists interested in second-messenger signal transduction and much has been learned about differences in the cellular localization and function of its different isotypes. In this study we systematically address the genomic locations and gene structures of the human PKC gene module. Results We first carried out fine chromosomal mapping of all nine PKC genes by fluorescence in situ hybridization (FISH), using cosmid and BAC probes. The PKC genes are found to be dispersed throughout the genome, and in some positions distinct from those previously reported: PKCα is at 17q24, PKCβ at 16p12, PKCγ at 19q13.4, PKCδ at 3p21.2, PKCε at 2p21, PKCζ at 1p36.3, PKCη at 14q22-23, PKCθ at 10p15 and PKCι at 3q26. For PKCι, an additional FISH signal mapped on Xq21.3 revealed a pseudogene (derived by retrotransposition). PKCγ, ζ, and θ are found to map to the most distal positions on the chromosomes, potentially implicating telomere position effects in their expression. Using the complete human genome draft sequence and bioinformatics tools, we then carried out a systematic analysis of PKC gene structure, including determination of the occurrence of single-nucleotide polymorphisms corresponding to the PKC loci. Conclusion This resource of genomic information now facilitates investigation of the PKC gene module in structural chromosomal abnormalities and human disease locus mapping studies.

Background Blood group genotyping is increasingly utilized for prenatal diagnosis and after recent transfusions, but still lacks the specificity of serology. In whites, the presence of antigen D is predicted, if two or more properly selected RHD-specific polymorphism are detected. This prediction must fail, if an antigen D negative RHD positive allele is encountered. Excluding RHDψ and CdeS frequent only in individuals of African descent, most of these alleles are unknown and the population frequency of any such allele has not been determined. Methods We screened 8,442 antigen D negative blood donations by RHD PCR-SSP. RHD PCR positive samples were further characterized by RHD exon specific PCR-SSP or sequencing. The phenotype of the identified alleles was checked and their frequencies in Germans were determined. Results We detected 50 RHD positive samples. Fifteen samples harbored one of three new Del alleles. Thirty samples were due to 14 different D negative alleles, only 5 of which were previously known. Nine of the 14 alleles may have been generated by gene conversion in cis, for which we proposed a mechanism triggered by hairpin formation of chromosomal DNA. The cumulative population frequency of the 14 D negative alleles was 1:1,500. Five samples represented a D+/- chimera, a weak D and three partial D, which had been missed by routine serology; two recipients transfused with blood of the D+/- chimera donor became anti-D immunized. Conclusion The results of this study allowed to devise an improved RHD genotyping strategy, the false-positive rate of which was lower than 1:10,000. The number of characterized RHD positive antigen D negative and Del alleles was more than doubled and their population frequencies in Europe were defined.

Systemic lupus erythematosus is the prototype multisystem autoimmune disease. A strong genetic component of susceptibility to the disease is well established. Studies of murine models of systemic lupus erythematosus have shown complex genetic interactions that influence both susceptibility and phenotypic expression. These models strongly suggest that several defects in similar pathways, e.g. clearance of immune complexes and/or apoptotic cell debris, can all result in disease expression. Studies in humans have found linkage to several overlapping regions on chromosome 1q, although the precise susceptibility gene or genes in these regions have yet to be identified. Recent studies of candidate genes, including Fcγ receptors, IL-6, and tumour necrosis factor-α, suggest that in human disease, genetic factors do play a role in disease susceptibility and clinical phenotype. The precise gene or genes involved and the strength of their influence do, however, appear to differ considerably in different populations.

Background Mutations in the CHK2 gene at chromosome 22q12.1 have been reported in families with Li-Fraumeni syndrome. Chk2 is an effector kinase that is activated in response to DNA damage and is involved in cell-cycle pathways and p53 pathways. Methods We screened 139 breast tumors for loss of heterozygosity at chromosome 22q, using seven microsatellite markers, and screened 119 breast tumors with single-strand conformation polymorphism and DNA sequencing for mutations in the CHK2 gene. Results Seventy-four of 139 sporadic breast tumors (53%) show loss of heterozygosity with at least one marker. These samples and 45 tumors from individuals carrying the BRCA2 999del5 mutation were screened for mutations in the CHK2 gene. In addition to putative polymorphic regions in short mononucleotide repeats in a non-coding exon and intron 2, a germ line variant (T59K) in the first coding exon was detected. On screening 1172 cancer patients for the T59K sequence variant, it was detected in a total of four breast-cancer patients, two colon-cancer patients, one stomach-cancer patient and one ovary-cancer patient, but not in 452 healthy individuals. A tumor-specific 5' splice site mutation at site +3 in intron 8 (TTgt [a → c]atg) was also detected. Conclusion We conclude that somatic CHK2 mutations are rare in breast cancer, but our results suggest a tumor suppressor function for CHK2 in a small proportion of breast tumors. Furthermore, our results suggest that the T59K CHK2 sequence variant is a low-penetrance allele with respect to tumor growth.

Comparative genomics provides at least three methods for identifying functional links between genes: examination of phylogenetic distributions, analysis of conserved proximity and observations of fusions of genes into a multidomain gene in another organism. We show that the functional networks obtained by applying these methods have different topologies and that the information they provide is largely additive. In particular, the combined networks of functional links contain an average of 57% of an organism's complete genetic complement, uncover substantial portions of known pathways, and suggest the function of previously unannotated genes. In addition, the combined networks are qualitatively different from the networks obtained using individual methods.

Background Celiac disease has a strong genetic association with HLA. However, this association only explains approximately half of the sibling risk for celiac disease. Therefore, other genes must be involved in susceptibility to celiac disease. We tested for linkage to genes or loci that could play a role in pathogenesis of celiac disease. Methods DNA samples, from members of 62 families with a minimum of two cases of celiac disease, were genotyped at HLA and at 13 candidate gene regions, including CD4, CTLA4, four T-cell receptor regions, and 7 insulin-dependent diabetes regions. Two-point and multipoint heterogeneity LOD (HLOD) scores were examined. Results The highest two-point and multipoint HLOD scores were obtained in the HLA region, with a two-point HLOD of 3.1 and a multipoint HLOD of 5.0. For the candidate genes, we found no evidence for linkage. Conclusions Our significant evidence of linkage to HLA replicates the known linkage and association of HLA with CD. In our families, likely candidate genes did not explain the susceptibility to celiac disease.

Background Since contradictory results have been reported, we reanalysed the 77C→G transition in exon 4 of the protein-tyrosine phosphatase receptor-type C (PTPRC also known as CD45) in a large cohort of German MS patients and controls. Different isoforms of the protein are expressed, depending on alternative splicing of exons 4 (CD45RA), 5 (CD45RB) and 6 (CD45RC) (CD45RO, exons 4–6 spliced out). The 77C→G transition does not change the amino acid sequence, but it is probably part of a motif necessary for splicing leading to the isoform CD45RA. The expression of CD45RA is increased in 77C/G heterozygous individuals. The aim of the study was to clarify the importance of the PTPRC 77C→G transition in our German cohort of MS patients. Methods PCR products of exon 4 were digested using endonuclease MspI. The resulting restriction fragments of the wildtype C allele are 198 and 62 bp in length. In the G allele an additional restriction site is present yielding fragments of 114 and 84 bp. Results The G allele was identified in 10 of the 347 controls (1.4%) and in 7 of 454 MS patients (0.8%; Table 1). No homozygous individuals were found either in the control or in the patient group. Genetic association between the PTPRC 77C→G transition and MS susceptibility was excluded in the MS cohort. In addition, subgrouping patients according to differences in the clinical course of MS or according to HLA-DRB1*15 status did not yield significant differences. Conclusions The 77C→G transition in exon 4 of the PTPRC gene may contribute to MS susceptibility only in very few families, if at all, but it is not relevant for the majority of MS cases, including virtually all German patients.

Background Prior to this report, members of the inward rectifier family, or Kir, have been found only in eukaryotes. Like most K+ channels, the pore-forming part of the protein is formed by four identical, or closely related, subunits. Each subunit contains a transmembrane M1-P-M2 motif that is followed by a relatively large C-terminus region unique to Kir's. Results In searching unfinished microbial genomes for K+ channels, we identified five sequences in the prokaryote Burkholderia pseudomallei, Burkholderia cepacia, Burkholderia fungorum LB400, Magentospirillum magnetotacticum, and Nostoc Punctiforme genomes that code for proteins whose closest relatives in current sequence databases are eukaryote Kir's. The sequence similarity includes the C-terminus portion of Kir's, for which there are no other close homologs in current prokaryote sequences. Sequences of the pore-forming P and M2 segments of these proteins, which we call KirBac, is intermediate between those of eukaryotic Kir's and several other K+ channel families. Conclusions Although KirBac's are more closely related to Kir's than to other families of K+ channels, the intermediate nature of their pore-forming P and M2 segments suggests that they resemble an ancestral precursor to the eukaryotic Kir's. The similarity of KirBac to the bacterial KcsA channel, whose transmembrane structure has been solved, helps align Kir's with KcsA. KirBac's may assist in solving the three-dimensional structure of a member of the Kir family since bacterial membrane proteins are more easily expressed in the quantities necessary for crystallography.

In rheumatic diseases, autoantibody-producing cells of interest are often hidden in a polyclonal B-lymphocyte population. Immunoglobulin gene fingerprinting is a useful approach to screen for expanding clones and to detect recirculation between different locations. The gene fingerprinting approach and the Southern blot technique have been amalgamated, using electrophoretic transfer of a PCR product from an acrylamide gel onto a nylon membrane followed by hybridization with specific oligonucleotide probes. In contrast to conventional fingerprinting, the authenticity of immunoglobulin genes can be confirmed, individual genes can be detected and handling radionucleotides can be avoided. Also, the membrane may be reused for further investigations.